The artificial humic substance HS1500 does not inhibit photosynthesis of the green alga Desmodesmus armatus in vivo but interacts with the photosynthetic apparatus of isolated spinach thylakoids in vitro.

Humic substances (HSs) can influence the growth and composition of freshwater phytoplankton assemblage. Since HSs contain many phenolic and quinonic moieties and cause growth reductions in eco-physiological field experiments, HSs are considered photosystem II herbicides. To test this specific mode of action in vivo and in vitro, respectively, we used intact cells of the green alga Desmodesmus armatus, as well as thylakoids isolated from spinach (Spinacia oleracea) as a model system for the green algal chloroplast. Photosynthetic electron transport was measured as oxygen evolution and variable chlorophyll fluorescence. The in vivo effect of the artificial humic substance HS1500 on algae consisted of no impact on photosynthesis-irradiance curves of intact green algae compared to untreated controls. In contrast, addition of HS1500 to isolated thylakoids resulted in light-induced oxygen consumption (Mehler reaction) as an in vitro effect. Fluorescence induction kinetics of HS-treated thylakoids revealed a large static quenching effect of HS1500, but no inhibitory effect on electron transport. For the case of intact algal cells, we conclude that the highly hydrophilic and rather large molecules of HS1500 are not taken up in effective quantities and, therefore, cannot interfere with photosynthesis. The in vitro tests show that HS1500 has no inhibitory effect on photosystem II but operates as a weak, oxygen-consuming Hill acceptor at photosystem I. Hence, the results indicate that eco-physiological field experiments should focus more strongly on effects of HSs on extracellular features, such as reducing and red-shifting the underwater light field or influencing nutrient availability by cation exchange within the plankton network.

Chlamydomonas reinhardtii mutants deficient for Old Yellow Enzyme 3 exhibit increased photooxidative stress.

Old Yellow Enzymes (OYEs) are flavin-containing ene-reductases that have been intensely studied with regard to their biotechnological potential for sustainable chemical syntheses. OYE-encoding genes are found throughout the domains of life, but their physiological role is mostly unknown, one reason for this being the promiscuity of most ene-reductases studied to date. The unicellular green alga Chlamydomonas reinhardtii possesses four genes coding for OYEs, three of which we have analyzed biochemically before. Ene-reductase CrOYE3 stood out in that it showed an unusually narrow substrate scope and converted N-methylmaleimide (NMI) with high rates. This was recapitulated in a C. reinhardtii croye3 mutant that, in contrast to the wild type, hardly degraded externally added NMI. Here we show that CrOYE3-mediated NMI conversion depends on electrons generated photosynthetically by photosystem II (PSII) and that the croye3 mutant exhibits slightly decreased photochemical quenching in high light. Non-photochemical quenching is strongly impaired in this mutant, and it shows enhanced oxidative stress. The phenotypes of the mutant suggest that C. reinhardtii CrOYE3 is involved in the protection against photooxidative stress, possibly by converting reactive carbonyl species derived from lipid peroxides or maleimides from tetrapyrrole degradation.

Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms.

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

A rapid and easy to handle thermoluminescence based technique for evaluation of carbon tetrachloride-induced oxidative stress on rat hepatocytes.

Oxidative stress has become one of the most intensively studied topics in biomedical research and is an often observed mechanism of non-genotoxic carcinogens like carbon tetrachloride. To monitor the oxidative stress status in in vitro hepatocytes, we compared thermoluminescence (TL) measurements with biochemical standard methods for oxidative stress markers. In contrast to biochemical analysis, TL measurements can be performed without any time-consuming extraction procedures by using directly collected cell material. After incubation with CCl(4) (24 h), thermo-induced light emission increased with rising concentration of CCl(4) up to eightfold at 10 mM CCl(4). Simultaneously, we determined the content of different secondary oxidative stress products, like thiobarbituric acid reactive substances and malondialdehyde. The rise of all biochemical markers complied with the increasing concentration of CCl(4). Finally, we could show that the CCl(4)-induced increase of oxidative stress markers determined by time-consuming biochemical methods perfectly correlates with the increase of high temperature bands in rapid TL measurements.

Light emission originating from photosystem II radical pair recombination is sensitive to zeaxanthin related non-photochemical quenching (NPQ).

We have used chlorophyll fluorescence, delayed luminescence and thermoluminescence measurements to study the influence of an artificial DeltapH in the presence or absence of zeaxanthin on photosystem II reactions. Energization of the pea thylakoid membranes induced non-photochemical fluorescence quenching and an increase in the overall luminescence emission of PSII during delayed luminescence and thermoluminescence measurements. This DeltapH-induced overall luminescence increase was caused by a strongly enhanced delayed luminescence in the seconds range before sample heating. In the subsequent thermoluminescence measurements the intensity of the B-band decreased after one and increased after two or more single turnover flashes. We propose that strong membrane energization shifted the redox potential of photosystem II radical pairs to more negative values causing the high delayed luminescence. The zeaxanthin-dependent non-photochemical fluorescence quenching component, however, did not alter thermoluminescence B-bands but decreased the delayed luminescence intensity by 30%. To our knowledge this is the first report that the radiative radical pair recombination, exhibited as delayed luminescence but not thermoluminescence emission, is sensitive to the antenna located zeaxanthin related non-photochemical fluorescence quenching. Our data can be interpreted within the frame of the exciton/radical pair equilibrium model that describes photosystem II as a shallow trap and incorporates the transfer of energy from the re-excitated reaction centre to the antenna of photosystem II.

Zwitterionic oligonucleotides: a study on binding properties of 2'-O-aminohexyl modifications.

2'-O-Aminohexyl side chains provide excellent conditions for zwitterionic interstrand and intrastrand interactions of oligonucleotides. 2'-O-Aminoalkylated phosphoramidites of adenosine and uridine were synthesized and incorporated in increasing number into homo adenosine and homo uridine/thymidine dodecamers, respectively. CD spectra of these dodecamers with complementary sense DNA exhibited a B-DNA type structure. While duplex stability values of all tested oligonucleotides were lower than those of the native oligonucleotides, they were significantly higher than those of 2'-O-heptyl modified oligonucleotides. The destabilization amounted to 0.9, 1.5, and 2.7 degrees C per modification for 2'-O-aminohexyl adenosine, 2'-O-aminohexyl uridine, and 2'-O-heptyl adenosine substitutions. These findings are pointing to a duplex stabilizing effect of the interaction of side chain amino groups with backbone phosphoric acid.

Longitudinal leaf gradients of UV-absorbing screening pigments in barley (Hordeum vulgare).

In vivo leaf characteristics were examined to describe longitudinal gradients of UV-absorbing screening pigments in barley. Chlorophyll fluorescence properties and in vivo absorption spectra (210-750 nm) of leaves were measured from the base to the tip. Barley leaves showed strong longitudinal gradients of chlorophyll, where chlorophyll concentration increased within the first 5-8 cm from the leaf base, and did not significantly change for the remaining part of the leaf. Fluorescence microscopy was used to localize cell wall bound screening pigments different from flavonoids, since flavonoids lack a blue-green fluorescence emission (Lichtenthaler and Schweiger 1998). Measurements of in vivo chlorophyll fluorescence indicated that the ratio of UV-absorbing screening pigments per leaf area increases from the leaf base to the tip. These gradients were confirmed by in vivo absorption spectra. It is demonstrated that leaves in the early stage of development are less protected against UV-radiation than fully developed mature leaf regions. The experiments show that measurements of in vivo chlorophyll fluorescence are ideally suited as a fast non-invasive tool to estimate the epidermal UV-transmittance in different leaf sections.

A new type of thermoluminometer: a highly sensitive tool in applied photosynthesis research and plant stress physiology.

Here we describe a newly developed thermoluminescence measuring device that employs flash excitation, peltier heating, and light detection by channel photomultipliers (CPM). The new thermoluminometer is equipped with four sample holders for simultaneous measurements of thermoinduced light emission in the temperature range from -20 degrees C to +180 degrees C. It allows one to measure leaf samples, chloroplasts, thylakoids, algae, or even bioorganic material lacking chlorophyll by means of naturally induced or artificially applied chemilumigenic probes. The temperature range of the thermoluminometer allows one to analyse the thermoinduced radical pair recombination of photosystem II in the lower temperature region as well as chemiluminescence from lipid peroxidation in the higher temperature region. Hence, plant material can be assessed concerning both its photosynthetic and its oxidative stress status. Since the device is equipped with four sample holders and four CPM channels for simultaneous detection of thermoinduced light emission, it facilitates a high throughput. Therefore, the new device is interesting, not only in ecophysiology, but also in the field of plant breeding, as it can be used to study the stress tolerance of various cultivars of cultural crop plants.

Thermoluminescence as a tool for monitoring ozone-stressed plants.

The effect of ozone (6 h, various concentrations from 0 to 350 ppb) on barley (Hordeum vulgare L., cv. Bomi) and tomato (Lycopersicon esculentum L., cv. Yellow Cherry) leaves was investigated in parallel by thermoluminescence (TL) and fluorescence (FL) methods. Several significant changes were found in TL glow curves measured after excitation by one single turnover flash at +2 degree C in the temperature range from 2 to 170 degree C immediately after ozone exposure. Contrary to TL, ozone induced only negligible changes in FL parameters F0, FM and Fv/FM. Measurements done 24 h after ozone exposure showed partial recovery of ozone-induced changes. The extent of recovery was not the same in different parts of TL curves. Fluorescence parameters were not significantly changed. The results demonstrate that TL parameters are more sensitive to ozone than conventially used FL parameters F0, FM and Fv/FM. Moreover, TL measurements seem to give information not only about the PSII electron transport, but also about the extent of oxidative damage and membrane lipid peroxidation. It is concluded, that TL can be a highly informative tool for monitoring the impact of ozone on plants.

Intra- and interspecific differences of 10 barley and 10 tomato cultivars in response to short-time UV-B radiation: a study analysing thermoluminescence, fluorescence, gas-exchange and biochemical parameters.

The impact of UV-B radiation on 10 genotypically different barley and tomato cultivars was tested in a predictive study to screen for potentially UV-tolerant accessions and to analyze underlying mechanisms for UV-B sensitivity. Plant response was analyzed by measuring thermoluminescence, fluorescence, gas exchange and antioxidant status. Generally, barley cultivars proved to be much more sensitive against UV-B radiation than tomato cultivars. Statistical cluster analysis could resolve two barley groups with distinct differences in reaction patterns. The UV-B sensitive group showed a stronger loss in PSII photochemistry and a lower gas-exchange performance and regulation after UV-B radiation compared to the more tolerant group. The results indicate that photosynthetic light and dark reactions have to play optimally in concert to render plants more tolerant against UV-B radiation. Hence, measuring thermoluminescence/fluorescence and gas exchange in parallel will have much higher potential in identifying tolerant cultivars and will help to understand the underlying mechanisms.

Evaluation of carbon tetrachloride-induced stress on rat hepatocytes by 31P NMR and MALDI-TOF mass spectrometry: lysophosphatidylcholine generation from unsaturated phosphatidylcholines.

Carbon tetrachloride (CCl(4)) represents an excellent model to study oxidative injury of cells. It is widely accepted that hepatocellular injury is a consequence of the metabolic conversion of CCl(4) into highly reactive, free radical intermediates. Among the direct toxic effects of CCl(4), stimulation of lipid peroxidation and the binding of the electrophilic radicals to membrane lipids have been suggested to play important roles in the pathogenesis of irreversible cell damage. CCl(4)-induced liver damage was modeled in cultures of rat hepatocytes with the focus on alterations of phosphatidylcholine (PC). The PC acyl chain composition was analyzed by (31)P NMR spectroscopy and MALDI-TOF mass spectrometry. The content of the membrane arachidonoyl PC was decreased by almost 30% after incubation of the cells with CCl(4). This relative decrease was found to correlate with increased concentrations of the corresponding saturated lysophosphatidylcholine (LPC). It is concluded that LPC represents a useful biomarker of CCl(4)-mediated damaging of hepatocytes. It is also speculated that de novo biosynthesis of PC is influenced by CCl(4).

2'-O-Lysylaminohexyl oligonucleotides: modifications for antisense and siRNA.

A novel type of oligonucleotide has been developed, characterized by the attachment of a lysyl moiety to a 2'-O-aminohexyl linker. A protected lysine building block was tethered to 2'-O-aminohexyluridine, and the product was converted into the corresponding phosphoramidite. Up to six modified nucleosides were incorporated in dodecamer DNA and RNA oligonucleotides using standard phosphoramidite chemistry. Each of the building blocks contributes one positive charge to the oligonucleotide instead of the negative charge of a wild-type nucleotide. Thermal denaturation profiles indicated a stabilizing effect of 2'-O-lysylaminohexyl chains that was more pronounced in RNA duplexes. Incubation of the oligonucleotides with 5'-exonuclease revealed an exceptionally high stability against enzymatic degradation. Incorporation of up to three modifications into functional antisense and siRNA oligonucleotides targeted at ICAM-1 showed that the gene-silencing activity was higher with an increasing number of lysylaminohexyl nucleotides. Compared with wild-type antisense or siRNA, compounds with three modifications led to equal or higher ICAM-1 downregulation.

Dual role of the plastid terminal oxidase in tomato.

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

Effects of UV irradiation on barley and tomato leaves: thermoluminescence as a method to screen the impact of UV radiation on crop plants.

The effect of different UV intensities and irradiation times on barley and tomato leaves was investigated by analysis of thermoluminescence (TL) and chlorophyll (chl) fluorescence measurements. Epifluorescence microscopy was used to estimate the epidermal UV transmittance of leaves. In barley a strong supression of TL emission from the S2QB- (B-band) and the S2QA- (Q-band) charge recombination was observed increasing with prolonged UV exposure. Primary barley leaves were more sensitive to UV than secondary leaves. In tomato plants a decrease in the B-band only takes place at very high UV intensities and after prolonged exposure times (4 h). The impact of UV in cotyledons was more pronounced than in pinnate leaves of tomato plants. The strong differences in sensitivity to UV in the investigated barley and tomato variety may be due to different concentrations of UV screening pigments in the epidermal layer as demonstrated by epifluorescence measurements. The results show that TL has the same potential to analyse the sensitivity or tolerance of crop plants to UV irradiation as routine fluorescence techniques. Furthermore, TL is directly monitoring the radical pair states of PSII and can distinguish between UV-induced donor and acceptor site-related damage.